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“Round up the usual suspects” A Comment on Nonexistent Plant GPCRs

Daisuke Urano and Alan M. Jones The Departments of Biology and Pharmacology, University of North Carolina, Chapel Hill, NC

37599-3280

In the classic 1942 movie Casablanca, Vichy Police Captain Louis Renault obfuscated

the truth by commanding his lieutenants to “round up the usual suspects” knowing well

that the culprit with the gun stood in plain view (Curtiz, 1942). Something similar has

happened in the plant G protein field. This Scientific Correspondence was written to

shed light on the source of misunderstanding and to pre-empt further confusion. Plant

heterotrimeric G proteins are self-activating and therefore do not need and do not utilize

G protein-coupled receptors (GPCR). This conclusion was reached previously from

biochemical analyses of plant G proteins (Johnston et al., 2007; Urano et al., 2012);

here, we buttress this point of view using an evolutionary argument. Proteins suspected

as plant GPCRs were “rounded up” because they have predicted topology of animal

GPCRs and/or have been mis-annotated as such, however these proteins are highly

conserved in organisms that lack heterotrimeric G proteins. Therefore they have

functions unrelated to G-coupled signaling. Instead, the culprit protein standing in plain

view is a receptor GTPase-accelerating protein (GAP), a receptor-GAP called AtRGS1.

GPCRs are Receptor-GEFs In animals and fungi, GPCRs are cell surface receptors that perceive a wide spectrum

of signals. The human genome encodes about 850 characterized plus candidate

(orphan) GPCRs, which constitute the largest human gene family (Nordström et al.,

2011). They are involved in the perception of various external signals, like light,

neurotransmitters or peptide hormones/pheromones, even proteolytic activity. As the

name designates, GPCRs are coupled to a cytoplasmic, membrane tethered,

heterotrimeric GTP-binding complex comprised of a Gα subunit partnered to an obligate

Gβγ dimer. Gα tightly binds GDP in the heterotrimeric deactivated complex. Upon

Plant Physiology Preview. Published on January 10, 2013, as DOI:10.1104/pp.112.212324

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receptor activation by its signal, the GPCR pries away the GDP from Gα and stabilizes

the open state of the complex (; Chung et al., 2011; Rasmussen et al., 2011) allowing

GTP, which is in excess over GDP in animal cells, to bind Gα (Schneider and Seifert,

2010). Thus, GPCRs should be considered as receptor enzymes having a Guanine

Nucleotide Exchange Factor (GEF) activity. In other words, GPCRs are receptor-GEFs.

The GTP-bound form of Gα changes its conformation to activate downstream effectors

that consequently alter cell behavior. Subsequently, the intrinsic GTP hydrolysis

property of Gα subunit returns Gα to the inactive GDP-bound form allowing re-

association of the heterotrimer, the resting state of this complex. This hydrolysis “back

reaction” is often stimulated by a GTPase Accelerating Protein (GAP) known as a

Regulator of G Signaling (RGS) protein.

The defining topological feature of GPCRs is the 7-transmembrane-spanning

(7TM) domain and it is this topological feature that is its most conserved feature.

However, conservation at the amino acid sequence level is poor among GPCRs, even

within a single species. Unfortunately, the 7TM topology alone is often used as

evidence to annotate divergent 7TM-encoding genes as GPCRs, although discussed

below, methods that do not rely on sequence alignments were used to assemble 7TM

receptor candidates (Moriyama and Kim, 2006; Moriyama et al., 2006; Gookin et al.,

2008; Lu et al., 2009)

The definitive test for a GPCR is GEF activity but GEF activity has been

demonstrated for only well-studied GPCRs. This is an onerous criterion for

classification that has relaxed over time and with the deluge of new genomes to

annotate.

Plant G proteins are self-activating.

In 2007, Francis Willard and colleagues (Johnston et al., 2007) showed that the

Arabidopsis Gα subunit (AtGPA1) is the fastest known nucleotide exchanging G protein,

having an astonishingly 200 times faster rate than the typical animal Gα subunit (Jones

et al., 2011; Jones et al., 2011; Jones et al., 2012). The spontaneous exchange

property of AtGPA1 paired with its slow GTPase property indicates that AtGPA1 is 99%

occupied by GTP in vitro. Evidence supports the conclusion that all plant Gα subunits



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spontaneously load GTP (Urano et al., 2012) and while this cycling property means that

nearly all Gα is in its active form in the test tube, that is not the case in planta.

Increasing the amount of GTP-bound AtGPA1 in plant cells confers “active” phenotypes

in vivo (Ullah et al., 2001; Chen et al., 2003). This means that the GTP-bound form is

the active Gα form in plant cells, just as it is in animal cells, and that an unknown

element in plant cells must be controlling this active state. That element is not a GPCR.

Not all 7TM proteins are GPCRs Possession of a 7TM domain does not justify a GPCR moniker. The insect odorant

receptors, originally discussed as GPCRs, are ligand-gated cation channels with the

amino-terminus inside the cell (Sato et al., 2008; Wicher et al., 2008). Further examples

are the green algae light sensor, which is homologous to bacteriorhodopsin and

functions as a light-activated channel (Nagel et al., 2002), the microbial type-I rhodopsin

functions as an ion pump (Oesterhelt and Stoeckenius, 1971), the human and fungal

adiponectin receptors have ceramidase activity (Kupchak et al., 2009; Villa et al., 2009),

and the bacterial homologue, hemolysin III has hemolysis activity (Baida and Kuzmin,

1996). In addition, some human genes annotated as orphan receptors are likely not

GPCRs. Notably, the human GPR89 (NP_001091081), GPR107 (NP_001130029),

GPR108 (NP_001073921), adiponectin receptors (NP_057083 and NP_078827) and

GPR175 (NP_001129525) have no sequence similarity to any characterized-GPCRs

(Tang et al., 2005; Nordström et al., 2011), and no evidence that they function as

GPCRs. As will be discussed later, plant proteins with homologies to these faux GPCRs

discussed above and those with predicted 7TMs are still annotated as candidate

GPCRs in the databases. Mis-annotation is one source of the plant GPCR problem.

Plants lack GPCRs with animal and fungal homology Mining genomes for divergent GPCRs is a daunting, if not impossible, task because

GPCRs evolved at a rapid pace (Fredriksson and Schiöth, 2005). Therefore, in 2006 as

a fresh approach to solve this problem, Etsuko Moriyama and colleagues avoided

comparing sequences and used nonconventional algorithms (Hill et al., 2002) to



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assemble a set of 54 candidate Arabidopsis 7TM receptors (Moriyama et al., 2006).

Two years later, this work was extended to rice proteins (Gookin et al., 2008). Included

in this set, are GCR1 (G PROTEIN COUPLED RECEPTOR 1), 15 MLO (MILDEW

RESISTANCE LOCUS O), 5 HHP (HEPTAHELICAL PROTEIN), AtRGS1 (Arabidopsis

REGULATOR OF G SIGNALING), TOM (Tobamovirus replication protein) and CAND

proteins (Candidate GPCR). None of these are plant GPCRs.

G-PROTEIN-COUPLED RECEPTOR 1 (GCR1). Except for GCR1, no plant

protein carries any vestige of GPCR homology. GCR1 homology to Dictyostelium cAR1

(cAMP Receptor 1) lies in the 3rd and 4th transmembrane spans and is weak at best.

Even if GCR1 is homologous to cAR1, it is still not clear whether cAR1, or at least the

ancestor of cAR1, was a GPCR. Furthermore, there is no biochemical proof that

Dictyostelium cAR1 has GEF activity, although there is indirect evidence showing

cAMP-induced FRET changes between Gα and Gβ subunits (Janetopoulos et al.,

2001). In lieu of direct biochemical proof that cAR1 is a receptor GEF, we turn to

evolution to assess its function. Slime mold’s cAR1 may be the closest extant protein to

the ancestor of animal GPCRs, but this ancestor was probably not a GPCR (Krishnan et

al., 2012). cAR1 is extant broadly in eukaryotes, notably found in alveolata, red algae,

and green algae but each of these groups lack a G protein signaling system therefore

the cAR1 homologs are not likely to activate G proteins (Fig. 1 and Supplemental Fig.

S1). Based on our argument that the cAR1 ancestor from which GCR1 evolved was not

a GPCR, we conclude that GCR1 does not activate G proteins.

Other reasons preclude GPCR functionality for GCR1 have been discussed

(Johnston et al., 2008). In addition, genetic epistasis shows that GCR1 and G proteins

act independently in at least some signaling pathways. GCR1 was reported to interact

physically with AtGPA1 but we have not been able to confirm that result (Huang and

Jones, unpublished) and deep screens for G protein and GCR1 partners have yet to

suggest a GCR1-Gα interaction.

MILDEW RESISTANCE LOCUS O (MLO). In 2002, Ralph Panstruga and

colleagues showed using loss-of-function mutations that the fungal resistance role for

MLO proteins is independent of G proteins (Kim et al., 2002). One might argue that this

finding does not exclude coupling to G proteins since the endogenous function of MLOs



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is unknown, however, the evidence to date does not suggest MLO proteins regulate the

activation state of G proteins. Epistasis analysis does not indicate that G proteins and

MLOs share the same signaling pathway, which is consistent with the conclusion that

MLOs are not coupled to G proteins. It should be noted that among the entire set of

candidate plant 7TM receptors, only barley MLO1 was confirmed biochemically to have

a 7TM domain (Devoto et al., 1999); as such, we emphasize that we are only refuting

the existence of plant GPCRs (receptor GEFs), not plant 7TM proteins.

HEPTAHELICAL PROTEIN 1-5 (HHP). HHPs were proposed as GPCR

candidates based on the similarity to human progestin and adipoQ receptors (PAQRs)

(Tang et al., 2005; Gookin et al., 2008). However, human PAQRs have no homology to

GPCRs (Tang et al., 2005), rather they have significant similarity to hemolysin III

(Pfam:PF03006) (Baida and Kuzmin, 1996) with a topology unlike GPCRs (Yamauchi et

al., 2003). While PAQRs stimulate inhibitory G protein pathways (Thomas et al., 2006;

Thomas et al., 2007; Thomas, 2008) they do so by acting as ceramidases (Kupchak et

al., 2009; Villa et al., 2009), which produce spingolipids (Moussatche and Lyons, 2012).

Sphingolipids are well-known ligands for GPCRs (Spiegel and Milstien, 2003) and

hence the root of this HHP confusion.

G-PROTEIN-COUPLED RECEPTOR 2 (GCR2) and GPCR-TYPE G PROTEINS. Although neither G-COUPLED RECEPTOR 2 (GCR2) nor GPCR-TYPE G

PROTEIN (GTG) were retrieved by 7TMR search engines, these proteins were

originally mis-annotated as Arabidopsis GPCRs in Gene Bank and accepted into the

plant biology community without a healthy dose of skepticism.

GCR2 shares sequence similarity with a cytoplasmic protein homologous to the

prokaryotic enzyme lanthionine synthase (Bauer et al., 2000; Mayer et al., 2001).

Despite that, Liu and coworkers proclaimed GCR2 to be a GPCR (Liu et al., 2007) and

subsequently the data from the original publication were quickly refuted (Gao et al.,

2007; Johnston et al., 2007; Chen and Ellis, 2008; Guo et al., 2008; Illingworth et al.,

2008).

Careful examination of topological predictions for GTG1 and 2 indicate these

proteins have 8 or 9 transmembrane domains; 8 would be consistent with the authors

own split-ubiquitin yeast complementation data (Pandey et al., 2009). GTGs are most



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likely Golgi ion transporters based on their homologous animal counterparts (Maeda et

al., 2008), consistent with GTG localization to the Golgi Apparatus (Jaffé et al., 2012).

CANDIDATE G-PROTEIN COUPLED RECEPTORs (CAND) and TOBAMOVIRUS REPLICATION PROTEIN 1 (TOM1) Gookin et al. reported several

other Arabidopsis GPCR candidates, including CAND proteins (Supplemental Figs. S4-

S6), unfortunately not to be confused with other Arabidopsis proteins of the prototype

abbreviation but a different name, Cullin-Associated and Neddylation-Dissociated

(Zhang et al., 2008). The authors reported interaction of several CAND proteins with

AtGPA1, using a yeast complementation assay (Gookin et al., 2008). CAND6 and

CAND7 are homologous to human GPR107 and GPR108 and CAND2 and CAND8 are

similar to human GPR175/TPRA40 (Vassilatis et al., 2003; Aki et al., 2008; Nordström

et al., 2011). Notably, these human proteins possess no sequence similarity to GPCRs

and are now classified into non-GPCR domain families (Fig. 1). These authors also

proposed that TOM1 and the distant homologues (CAND 3/4/5) were candidate plant

GPCRs (Supplemental Fig. S4), although there is no homology between these proteins

and GPCRs. TOM proteins have a domain of unknown function (DUF 1084) not found

in animal genomes.

The functions of candidate plant GPCRs predate the origin of G proteins.

Figure 1 shows the distribution of genes encoding G protein subunits (Gα, Gβ and Gγ)

and GPCR candidates. Gα, Gβ and Gγ genes are lacking within certain evolutionary

clades such as red and green algae and alveolata (Anantharaman et al., 2011). The

GPCR candidates described above are present in the clades lacking Gα, Gβ and Gγ

genes (Fig. 1 and Supplemental Figs. S1-S7). Under the neutral theory of molecular

evolution (Kimura, 1968), DNA sequences are mutated randomly and gradually lose the

original signature because there is no evolutionary pressure for synonymous mutations

to be restored to the original value (Nei, 2005). This is not the case for non-synonymous

mutations. For these, evolutionary constraint is not only applied by the intrinsic

molecular function (e.g. catalytic core residues of enzymes), but also by other

molecules (i.e. binding surfaces with ligands, proteins or DNA) (Temple et al., 2010).

For example, where we see that Gα, Gβ and Gγ subunits are independently deleted



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within certain evolutionary clades (Anantharaman et al., 2011), the loss as of the

collective group is correlated (Anantharaman et al., 2011). In other words, once a

genome loses one of the three subunits, there is little genetic constraint to keep the

other two genes. On the other hand, when proteins do not evolve rapidly after the loss

of a hypothesized protein partner, there is some other constraint. For example, proteins

like the candidate plant GPCRs discussed above did not evolve much in the absence of

G proteins (in certain organisms) indicating that these proteins have evolutionary

constraints that are unrelated to G signaling.

Plant G protein cycling The evidence indicates that regulation of plant G protein cycling is at the hydrolysis

step, not the nucleotide exchange step. That means either a GAP (i.e. an RGS protein)

or a GDP-dissociation protein (GDI) is regulating the active state of plant G proteins. A

GDI serving this job makes more sense. Assuming that GTP levels in plant cells are in

excess of GDP, uncontrolled consumption of GTP promoted by an RGS protein just to

keep G protein cycling in the inactive state is energy expensive. Logic dictates that

there must be a GDI, rather than a GAP, because GDIs simply “hold” the G protein in its

GDP-bound active state and do not promote nucleotide consumption as do the GAPs.

Another reason a GDI makes sense is that not all plants have RGS proteins; cereals

and some lower plants have self-activating G proteins but lack a canonical RGS protein

(Urano et al., 2012). For these species, we speculate a switchable (e.g. ligand-

regulated) GDI serves the purpose of regulating the plant G protein activation state.

“Louie, I think this is the beginning of a beautiful friendship.” The (mis)-annotation of a plant protein as a GPCR in a database prompts an irresistible

urge to order the mutants from the stock center, phenotype them, and submit the

dataset for a quick publication, all along riding on the coat-tails of Nobel Laureates who

discovered the original and bona fide GPCRs in animal cells. Similarly, obtaining a

topological prediction of a 7TM domain in a plant protein should not make us want to

“Play it again, Sam”. We simply point out that plants do not need and therefore do not

use animal-like GPCRs to control the active state of heterotrimeric G proteins. Instead

of embracing the animal GPCR paradigm, our collective research effort would be more



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productive if we focused on the mechanism of G cycle regulation in plants in the

absence of GPCRs.

Keywords: AtRGS1, cAR1, GCR1, GTG1, MLO, Plant GPCRs

Abbreviations: 7TM, seven-transmembrane; AtGPA1, Arabidopsis thaliana G Protein

subunit Alpha; AtRGS1, Arabidopsis thaliana Regulator of G Signaling protein 1; cAR1,

cAMP Receptor 1; CAND, Candidate GPCR; GAP, GTPase Accelerating Protein; GDI,

GDP dissociation inhibitor; GEF, Guanine nucleotide Exchange Factor; GPCR, G-

Protein Coupled Receptor; GTG, GPCR-Type G Protein; HHP, Heptahelical Protein;

MLO, Mildew Resistance Locus O; TOM, Tobamovirus Replication Protein

Acknowledgements: Work in the Jones Lab is supported by the NIGMS

(R01GM065989), NSF (MCB-0723515 and MCB-0718202), and The Division of

Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of

the US Department of Energy (DE-FG02-05er15671).

Figure legend

Figure 1. Gene conservation of G protein components and plant GPCR candidates

Genes homologous to Gα, Gβ, Gγ and plant GPCR candidates were identified as

mentioned in supplemental information. The Pfam domain was determined using

Arabidopsis genes shown on left of table. Color dots indicate gene conservations.

Phylogenetic trees for GPCR candidates are available in supplemental information. *

WD40 (PF00400) contains Gβ and other proteins possessing WD40 repeats. ** P.

sitchensis XLG is currently not registered in NCBI database, but found in EST data for

Picea glauca.



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